PROJECT SUMMARY Over 250,000 cases of breast cancer are diagnosed annually in the US alone, and about 80% of these will be estrogen receptor-? positive (ER+). Despite great progress in treating this ER+ disease by endocrine therapy, resistanceto this treatment remains a major problem, causing the majority of deaths from breast cancer. Our clinical studies on resistance have found that inactivation of the NF1 (for neurofibromatosis type 1) gene, which may occur in as much as 20% of primary ER+ breast cancer patients, correlates with a poor outcome in tamoxifen-treated patients. NF1 encodes neurofibromin, best known as a Ras repressor (a GTPase Activating Protein, or GAP), but the scie ntific premise of this project stems from our discovery that besides activating Ras, loss of NF1 also globally enhances estradiol (E2)-dependent gene expression, thus permitting the cells to grow in lower levels of E2 or even in tamoxifen, because NF1 also directly interacts with ER as a transcriptional co- repressor. In contrast to their lack of response to E2-deprivation or tamoxifen treatment, NF1-depleted cells still respond to fulvestrant, a SERD (Selective ER Degrader), and although the reduced repression of Ras-Raf signaling in NF1-depleted cells still allows promotion of cell survival/growth, this in turn could be blocked pharmacologically by FDA-approved kinase inhibitors. This project will therefore investigate the hypothesis that NF1-deficient ER+ breast tumors should be treated by a SERD together with inhibitors bloc king the Ras-Raf pathway. To assess whether NF1-status in patient tumors can differentiate treatment responses to various endocrine agents, Aim 1 will first establish an effective diagnosis for NF1-deficiency and then analyze how NF1- status impacts long-term treatment responses in two large randomized neoadjuvant clinical trials, ALTERNATE and P024. The former compares fulvestrant vs. AI (anastrozole), while the latter compares tamoxifen vs. another AI (letrozole). In addition, we propose that NF1-deficient ER+ breast cancer should be treated by a SERD combined with kinase inhibitors targeting the Ras-Raf pathway, so that Aim 2 will conduct preclinical modeling to examine new generation oral SERDs and Ras effector kinase inhibitors to find the best combination to drive clinical trial design and encourage pharma interest in trial support. A Phase -2 clinical trial to further this treatment concept has been planned, for which we already have pharma interest and commitment. We will support this trial here by developing technologies to assess how NF1-heterogeneity impacts treatment response. Finally, Aim 3 will analyze kinome reprograming after SERD treatment in primary NF1-deficient patients (Aim 1) and in preclinical models (Aim 2) by a mass spectrometry-based micro-scaled platform called KiP (Kinome Profiling). While our primary objective is to assess whether the use of a SERD in NF1 -depleted tumors can lead to compensatory Ras-Raf activation, the KiP platform is intrinsically unbiased and may also discover additional kinases driving SERD resistance. The successful execution of this project may stop the progression of the NF1- deficient subset of ER+ aggressive breast cancer early in its tracks.